Chemokines (chemotactic cytokines) act as molecular beacons for the recruitment and activation of T lymphocytes, neutrophils and macrophages, flagging pathogen battlegrounds. Recruitment of leukocytes, the white blood cells responsible for fighting infections depends on gradients of chemokines. Chemokines are a superfamily of small proteins (8-10 KD) that mediate diverse biological processes including leukocyte trafficking and homing, immunoregulation, hematopoiesis and antiogenesis. To date, 24 chemokine receptors are known. Chemokines play a fundamental role in innate immunity and inflammatory reactions (Baggiolini et al. (1994); Baggiolini et al. (1997); Rollins (1997).) Four subfamilies of chemokines have been described, based on the distance between the first two conserved cysteine residues: C, CC, CXC, and CX3C. All known chemokines signal through four groups of seven transmembrane receptors which belong to the G protein-coupled receptor and pertussis toxin-sensitive heterotrimeric G proteins of Gi family: XCR, CCR, CXCR and CX3CR. (Murphy et al. (2000)). Extracellular binding events can activate specific signal transduction pathways leading to various responses, such as chemotaxis. In the chemokine system, multiple chemokines may activate a single chemokine receptor; for example, the receptor CCR1 ligates the RANTES (regulated on activation normal T cell expressed), MIP-1α (macrophage inflammatory protein) and MIP-1β chemokines. Likewise, a single chemokine may activate several receptors (Mantovani (1999)).
Monocytes and neutrophils, which play an important role in the pathogenesis of inflammation and in antigen presentation, respond to chemokines (Lee et al. (2000)). Monocytes express the chemokine receptors CCR1, CCR2, CCR5, CCR8, CXCR2, and CXCR4. (Uguccioni et al. (1995); Weber et al. (2000)). The ligands MIP-1α and Monocyte Chemoattractant Protein 1 (MCP1) have been reported as potent monocyte activators in vitro. (Fantuzzi et al. (1999).) Neutrophils are crucial during many acute inflammatory responses, and may also play a role in orienting immunity toward Th1 responses. (Bonecchi et al. (1999).) They mainly respond to some CXC chemokines but do not migrate to most of CC chemokines. Human neutrophils express two high affinity IL-8 receptors, CXCR1 and CXCR2.
The chemokine CKβ8, also known as CCL23; hmrp-2a; myeloid progenitor inhibitor factor 1 (MPIF-1); SCYA23 (current nomenclature and Genome ID system), is a 99-amino acid CC chemokine containing six cysteines. It is constitutively expressed in liver, lung, pancreas, and bone marrow. CKβ8 has chemotactic activity on monocytes, dendritic cells, and resting lymphocytes (Forssmann et al. (1997)) and inhibits colony formation of bone marrow-derived low proliferative potential colony-forming cells. (Patel et al. (1997)). CKβ8-1, an alternative splicing form of CKβ8 that is 116-amino acids in length, has been reported. Both the CKβ8 and CKβ8-1 mature forms have been assigned as ligands for the CCR1 receptor. (Youn et al. (1998)). Cross-desensitization studies in both monocytes and eosinophils indicate that CKβ8-1 binds predominately to the CCR1. Further processing at the NH2-terminus of CKβ8 results in 76 or 75 residue proteins that are significantly more active on CCR1 expressing cells (Macphee et al. (1998), Berkhout et al. (2000)).
In addition to the chemokine receptors, neutrophils and monocytes also express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue N-formyl peptide receptor like 1 (FPRL1). Since the ligands for FPRL1 were unknown when it was originally cloned, FPRL1 was initially defined as an orphan receptor. (Bao et al. (1992); Murphy et al. (1992); Ye et al. (1992).) It was assigned as a LXA4 receptor since it binds lipoxin A4 (Fiore et al. (1994).) In addition, several different peptides/proteins have been reported to bind FPRL1 with low affinity (see FIG. 1). A serum amyloid A, a protein secreted during the acute phase of inflammation, has been reported as a medial affinity functional ligand (Su et al. (1999)). A β amyloid fragment (1-42) and neurotoxic prion peptide 106-126 are also low affinity ligands, indicating that FPRL1 may play a role in neurodegenerative diseases (Le et al. (2001)). Some other low affinity ligands include: peptides derived from HIV envelope proteins (Su et al. (1999), Deng et al. (1999)); and a Helicobacter pylori peptide, Hp(2-20). Some synthetic peptides, such as Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm) and Trp-Lys-Tyr-Met-Val-Met-NH2 (WKYMVM) (“W peptides 1 and 2”), have been reported as potent ligands for the receptor. (Christophe et al. (2001); Baek et al. (1996)). However, these non-naturally occurring peptides derived from random hexapeptide libraries have not been shown to be physiologically relevant.
In 1979, the World Health Organization announced that small pox had been vanquished—almost 200 years after the first small pox vaccination (puss from a cow pox-infected milkmaid) had been administered to a young boy, James Phipps. His life was spared from small pox infection because Edward Jenner had discovered that milkmaids that had contracted cow pox rarely catch small pox. The success of such a risky procedure was due to the molecular similarity of cow pox to small pox. Phipps' immune system could immediately mount a specific response upon the introduction of small pox, quickly disposing of the invaders.
Since then, many vaccines have been developed to prevent infection from a wide variety of agents, such as infectious microorganisms (bacteria and viruses), toxins, and even tumors. Despite significant advances since the 1790s, many infectious agents are free to prey on susceptible individuals because no effective vaccines exist. A glaring example, now having devastating quality-of-life and economic effects in many parts of the world is the human immunodeficiency virus (HIV). In the cases where vaccines do exist, they often are not available to those people and countries which lack access to funds, technical expertise and labor for multiple administrations. Any reduction in necessary resources, such as the number of required administrations to afford protection, would facilitate vaccination (immunization) of greater numbers of individuals.
Vaccination exploits the immune system, which comprises leukocytes (white blood cells (WBCs): T and B lymphocytes, monocytes, eosinophils, basophils, and neutrophils), lymphoid tissues and lymphoid vessels. To combat infection, B and T lymphocytes circulate throughout the body, interact with antigen-presenting cells and detect pathogens. Once an invader is detected, cytotoxic T cells or antibody-secreting B cells specific for the foreign agent are recruited to the infection site to destroy it. The concept of vaccination is to generate the same types of host-protective immune responses without exposing the individual to the pathology-inducing foreign agent (such as a pathogen or tumor). Such immune responses may be, for example, cell-mediated and/or antibody based.
Key player in the adaptive immune response to foreign invaders are the antigen presenting cells (APCs), such as macrophages, activated B cells and dendritic cells. Dendritic cells are especially important in the immune response. Immature or resting dendritic cells reside in epithelial layers, phagocytosing foreign material (called antigens). These dendritic cells become activated by tumor necrosis factor (TNF) secreted by nearby macrophages that have been stimulated by the foreign material. These activated dendritic cells, laden with foreign antigens, travel through the lymphatic system to the nearest lymph node. There, resting naive (unexposed to antigen) T cells whose antigen-specific receptors recognize the foreign antigen are activated, and the immune system is triggered into action.
While vaccination can be accomplished with attenuated or dead infectious agents, the safest vaccinations are those that provoke an immune response to a subset of isolated antigens or epitopes, expressed by the foreign agent. However, many such antigens are by themselves are weakly immunogenic or incompetent for instigating a strong immune response. To enhance the effectiveness of such antigens, adjuvants are often added to vaccine compositions. Examples of adjuvants include oil emulsions of dead mycobacteria (Freund's complete), other dead bacteria (e.g., B. pertussis), bacterial polysaccharides, bacterial heat-shock proteins or bacterial DNA. While effective, many of these adjuvants cause significant inflammation and are not suitable for human administration.
Present immunization methods are not effective for all antigens, for all individuals, or for eliciting all forms of protective immunity. In addition, the number of useful adjuvants is small and directed mainly to antibody-related immunity and not to cell-mediated immunity. Moreover, there is a considerable lag time from immunization until the immune system provides protection for the subject. Improved vaccine compositions and/or effective safe adjuvants capable of inducing cell-mediated responses as well as antibody, would greatly aid current vaccination efforts.